Stabilizing agents and capture ligands for use in assays measuring analyte concentrations

ABSTRACT

The present invention is related to compositions useful for the measurement of free or unbound analyte concentrations in a fluid. The present invention includes the use of capture ligands and stabilizing agents to improve the accuracy of analyte concentration assays. Methods and tools for using the present invention are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application60/833,786, filed Jul. 28, 2006, which is incorporated herein in itsentirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention teaches new and useful compositions for themeasurement of free or unbound analyte concentrations in a fluid. Thepresent invention includes the use of capture ligands and stabilizingagents to improve the accuracy of analyte concentration assays. Methodsand tools for using the present invention are also disclosed.

2. Background Art

Various assays have been developed to measure the concentration of ananalyte in solution. For example, assays have included mixing a solutioncontaining an analyte with a capture ligand, washing away anyimpurities, and then measuring the analyte associated with the captureligand. Several technical difficulties exist with these detectionmethods. In some instances, unintended binding of impurities to thecapture ligand occurs. Additionally, in some instances the binding ofthe analyte to the capture ligand is not stable and the analytedisassociates from the ligand. In some instances, the analyte in asolution can exist in a bound form (e.g., bound to a protein) and anunbound form, and it may be desirable to only measure the concentrationof the unbound form. Thus, in some instances it is necessary tostabilize the equilibrium between bound and unbound forms in order toobtain an accurate and reproducible measurement of analyteconcentration.

To address these problems, Beckman Coulter, Incorporated (BCI,Fullerton, Calif.) has traditionally used an alkyl aminefluoro-surfactant (FC100, manufactured by 3M Corporation, St. Paul,Minn.) in BCI's various analyte concentration assays. FC100 is a complexcocktail of amine fluoro-surfactants that exhibits substantial lot tolot variability in its exact chemical composition. According to 3MCorporation's Material Safety Data Sheet (MSDS, issued Feb. 8, 2000,Document Number 10-3799-3), FC100 contains water, diethylene glycolbutyl ether, fluoroalkyl sulfonate sodium salt, and a trade secretmixture of residual organic fluorochemicals. Without being limited bytheory, the inventors believe that an alkyl amine fluoro-surfactantfacilitates the accurate determination of the amount of free unboundanalyte versus bound analyte present in a sample solution by stabilizingthe equilibrium and therefore allowing for accurate measurement ofanalyte concentration.

Several years ago, the Environmental Protection Agency of the UnitedStates expressed concern regarding certain fluorocarbon compounds, suchas fluorocarbon octanoic acid and its derivatives, as potentiallyhazardous compounds. Therefore, a need exists for an alternative toreplace the alkyl amine fluoro-surfactant with a different assaycomposition that can function in determining analyte concentration.

The present invention provides novel compositions as well as methods andkits for using those compositions in assays to accurately measure thefree unbound analyte in a sample from a subject.

BRIEF SUMMARY OF THE INVENTION

The present invention addresses the needs mentioned above and theproblems encountered with currently available technologies.

The present invention is directed to methods for measuring aconcentration of free analyte, the methods comprising:

-   -   (a) adding a capture ligand to a vessel;    -   (b) adding a stabilizing agent to the vessel;    -   (c) adding a sample comprising the free analyte to the vessel;    -   (d) adding a detection system to the vessel; and    -   (e) measuring the concentration of the free analyte in the        sample using the detection system.

The present invention is also directed to compositions for use in anassay measuring the concentration of a free analyte, the compositionscomprising a capture ligand for the free analyte, and a stabilizingagent, with the proviso that the stabilizing agent does not comprise analkyl amine fluoro-surfactant.

The present invention is also directed to kits for use in estimating aconcentration of a free analyte, the kits comprising:

-   -   (a) a capture ligand for the analyte;    -   (b) a stabilizing agent; and    -   (c) a detection system.

In some embodiments, the kit can further comprise (d) a referencestandard.

The present invention is also directed to stabilizing agents comprisingthe general formula RX, wherein R and X are covalently bound, andwherein R is a saturated or unsaturated alkyl, alkoxy, alkoxyalkyl,alkoxyalkoxyalkyl, alkylaminoalkyl, alkylaminooxyalkyl,alkoxyaminoalkyl, phosphonoalkyl, carboxyalkyl, carboxyalkoxyalkyl,carboxyalkylaminoalkyl, carboxyalkylamidoalkyl, cycloalkyl,cycloalkylalkyl, hetercycloalkylalkyl, heterocyclyl, aryl, arylalkyl,heteroaryl, heteroarylalkyl, siloxane, or carbosilane, any of which canbe optionally interrupted with one or more oxygen, nitrogen,phosphorous, or silicon atoms, and wherein any one of the Rsubstitutents can be optionally substituted with one or more hydroxy,carbonyl, halogen, phosphate, acetyl, ammonium, or combinations thereof,and wherein X is sulfate, sulfonic acid, sulfonate, sulfite, taurate,sulfosuccinate, sulfobetaine, sulfonamide, methoxymethanilamide,sulfamyl, sulfeno, chlorite, chloride, iodide, iodate, bromide, bromate,fluoride, fluorate, nitrate, nitrite, nitroamine, amino, imino,isocyanoto, isothiocyano, acetamido, acetimido, azido, diazo, cyano,cyanato, phosphate, phospho, phosphono, phosphinyl, phosphino,carboxylate, acrylate, sebacate, phthalate, acetate, oxide, borate,peroxoborate, tetraborate, boranate, silane, orthosilcate, metasilicate,or a metal silicate, with the proviso that RX cannot be an alkyl aminefluoro-surfactant. FIGS. 1 and 2 describe stabilizing agents comprisingthe general formula RX.

In some embodiments, R can be alkyl, alkylene, alkyne, alkoxyalkyl,alkoxyalkoxyalkyl, alkylaminoalkyl, alkylaminooxyalkyl,alkoxyaminoalkyl, phosphonoalkyl, carboxyalkyl, carboxyalkoxyalkyl,wherein any one of which is optionally substituted with one or morehydroxy, carbonyl, halogen, phosphate, acetyl, ammonium, or combinationsthereof.

In some embodiments, R can be propyl, isopropyl, butyl, isobutyl,pentyl, hexyl, heptyl, octyl, 2-methyl butyl, 2-ethyl butyl, 2-propylbutyl, 3-methyl butyl, 3-ethyl butyl, 3-propyl pentyl, 2-methyl pentyl,2-ethyl pentyl, 2-propyl pentyl, 3-methyl pentyl, 3-ethyl pentyl,3-propyl pentyl, 4-methyl pentyl, 4-ethyl pentyl, 4-propyl pentyl,2-methyl hexyl, 2-ethyl hexyl, 2-propyl hexyl, 3-methyl hexyl, 3-ethylhexyl, 3-propyl hexyl, 4-methyl hexyl, 4-ethyl hexyl, 4-propyl hexyl,5-methyl hexyl, 5-ethyl hexyl, 5-propyl hexyl, 2-methyl heptyl, 2-ethylheptyl, 2-propyl heptyl, 3-methyl heptyl, 3-ethyl heptyl, 3-propylheptyl, 4-methyl heptyl, 4-ethyl heptyl, 4-propyl heptyl, 5-methylheptyl, 5-ethyl heptyl, 5-propyl heptyl, 6-methyl heptyl, 6-ethylheptyl, 6-propyl heptyl, 2-methyl octyl, 2-ethyl octyl, 2-propyl octyl,3-methyl octyl, 3-ethyl octyl, 3-propyl octyl, 4-methyl octyl, 4-ethyloctyl, 4-propyl octyl, 5-methyl octyl, 5-ethyl octyl, 5-propyl octyl,6-methyl octyl, 6-ethyl octyl, 6-propyl octyl, any of which can beoptionally substituted with one or more hydroxy, carbonyl, halogen,phosphate, acetyl, ammonium, or combinations thereof.

In some embodiments, X can be a sulfate, sulfonic acid, sulfonate,sulfite, taurate, sulfosuccinate, sulfobetaine, sulfonamide,methoxymethanilamide, sulfamyl, or sulfeno, preferably sulfate, sulfonicacid, sulfosuccinate, or taurate.

In some embodiments, R is C₆₋₁₈ alkyl and X is sulfate, sulfonate orsulfonic acid. In some embodiments, the stabilizing agent is selectedfrom the group consisting of 2-ethyl-hexyl sulfate; 1-hexane sulfonicacid, or salt thereof; 1-heptane sulfonic acid, or salt thereof;1-octane sulfonic acid, or salt thereof; 1-decane sulfonic acid, or saltthereof; sodium C₁₄₋₁₆ olefin sulfonate; sodium dodecyl sulfate; sodiumdioctyl sulfosuccinate; sodium N-oleyl-N-methyltaurate; sodiumpolyoxyethylene lauryl sulfate; amine alkylbenzyl sulfonate; sodiumethyl-hexyl sulfate; and combinations thereof. In some embodiments, thestabilizing agent is 2-ethyl-hexyl sulfate. In some embodiments, thecomposition comprises a salt of the stabilizing agent.

In some embodiments, the analyte is a hormone, drug, or vitamin. In someembodiments, the analyte comprises a single enantiomer or a de-iodinatedform of the analyte.

In some embodiments, the capture ligand and the stabilizing agent arecombined in a manner that maintains the equilibrium between free analyteand bound analyte in a sample to be analyzed.

In some embodiments, a volume fraction of the stabilizing agent is about0.0001 to about 0.1 volume percent.

In some embodiments, a concentration of the stabilizing agent is about 5to about 750 micromolar.

In some embodiments, a volume fraction of the 2-ethyl-hexyl sulfate isabout 0.004 to about 0.015 volume percent.

In some embodiments, a concentration of the 2-ethyl-hexyl sulfate isabout 75 to about 650 micromolar.

In some embodiments, the capture ligand is selected from the groupconsisting of an antibody, antibody fragment, antibody mimic, oranalyte-specific binding protein such as intrinsic factor orfolate-binding protein. In some embodiments, the capture ligand isimmobilized on a solid phase. In some embodiments, the capture ligand isan analyte-specific binding protein. In some embodiments, the captureligand is an antibody.

In some embodiments, the analyte is a thyroid hormone.

In some embodiments, the present invention further comprises animmunoassay system. In some embodiments, an immunoassay system comprisesparamagnetic particles coated with a biotin-specific binding molecule, abiotinylated analyte-specific capture protein, or a combination thereof.

In some embodiments, the immunoassay system comprises a detectablelabel.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a chart illustrating various R groups (I through VIII) forforming the stabilizing agents disclosed in the present invention.

FIG. 2 is a chart illustrating various X groups (I through VIII) forforming the stabilizing agents disclosed in the present invention.

FIG. 3A is a chart illustrating various RX compounds evaluated asstabilizing agents disclosed in the present invention. “Working conc V/V%” refers to the concentration of the stabilizing agent tested that wassoluble.

FIG. 3B is a chart illustrating various RX compounds evaluated asstabilizing agents disclosed in the present invention. “Working concV/V%” refers to the concentration of the stabilizing agent tested thatwas soluble.

FIG. 3C is a chart illustrating various RX compounds evaluated asstabilizing agents disclosed in the present invention. “Working concV/V%” refers to the concentration of the stabilizing agent tested thatwas soluble.

FIG. 3D is a chart illustrating various RX compounds evaluated asstabilizing agents disclosed in the present invention. “Working concV/V%” refers to the concentration of the stabilizing agent tested thatwas soluble.

FIG. 4 is a chart illustrating various EHS homologs evaluated asstabilizing agents disclosed in the present invention. “Working conc V/V%” refers to the concentration of the stabilizing agent tested that wassoluble.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a composition for use in an assaymeasuring the concentration of a free analyte, the compositioncomprising a capture ligand for the free analyte and a stabilizingagent, with the proviso that the stabilizing agent does not comprise analkyl amine fluoro-surfactant.

It is understood that as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include singular andplural references unless the context clearly dictates otherwise. Thus,for example, “a stabilizing agent” includes a single stabilizing agentas well as two or more different stabilizing agents in combination.

The term “optionally derivatized” refers to the subject molecule ormolecular moiety being optionally substituted, optionally interrupted,or both. The term “optionally substituted” refers to the replacement ofa hydrogen or carbon atom in a subject molecule or molecular moiety inexchange for an atom or group of atoms. The term “optionallyinterrupted” as described herein refers to the insertion of Si, O, N, S,or P into a backbone of a carbon chain or a siloxane chain. In someembodiments, the term “optionally derivatized” refers to thesubstitution or interruption with one or more oxygen, nitrogen, halogen,or a moiety containing an oxygen, nitrogen, or halogen.

The composition of the present invention can be used in an assaymeasuring the concentration of a free analyte. The composition cancomprise (i) a capture ligand for the free analyte; and (ii) astabilizing agent, wherein the stabilizing agent comprises the generalformula RX, or salt thereof, wherein R is a saturated or unsaturatedalkyl, aryl, or silicon-based polymer, any of which is optionallyderivatized with one or more moiety containing oxygen, nitrogen,halogen, or combination thereof, and wherein X is an oxoanion or oxideof sulfur or phosphorus, an oxoanion or carbonyl derivative of nitrogen,a carboxylate, a borate, a silicate, or a halogen, with the proviso thatthe stabilizing agent does not comprise an alkyl aminefluoro-surfactant.

In some embodiments, the stabilizing agent comprises the general formulaRX, or salt thereof, wherein R is a saturated or unsaturated alkyl,alkoxy, alkoxyalkyl, alkoxyalkoxyalkyl, alkylaminoalkyl,alkylaminooxyalkyl, alkoxyaminoalkyl, phosphonoalkyl, carboxyalkyl,carboxyalkoxyalkyl, carboxyalkylaminoalkyl, carboxyalkylamidoalkyl,cycloalkyl, cycloalkylalkyl, hetercycloalkylalkyl, heterocyclyl, aryl,arylalkyl, heteroaryl, heteroarylalkyl, siloxane, silicone, orcarbosilane, any of which can be optionally interrupted with one or moreoxygen, nitrogen, phosphorous, or silicon atoms, and wherein any one ofthe R substitutents can be optionally substituted with one or morehydroxy, carbonyl, halogen, phosphate, acetyl, ammonium, or combinationsthereof, and wherein X is sulfate, sulfonic acid, sulfonate, sulfite,taurate, sulfosuccinate, sulfobetaine, sulfonamide,methoxymethanilamide, sulfamyl, sulfeno, chlorite, chloride, iodide,iodate, bromide, bromate, fluoride, fluorate, nitrate, nitrite,nitroamine, amino, imino, isocyanoto, isothiocyano, acetamido,acetimido, azido, diazo, cyano, cyanato, phosphate, phospho, phosphono,phosphinyl, phosphino, carboxylate, acrylate, sebacate, phthalate,acetate, oxide, borate, peroxoborate, tetraborate, boranate, silane,orthosilcate, metasilicate, or a metal silicate with the proviso that RXis not an alkyl amine fluoro-surfactant, i.e., R is not a fluoro alkylamine.

FIGS. 1 and 2 outline various R and X groups of the stabilizing agent ofthe present invention. In some embodiments, the stabilizing agents maybe of a formula RX. In other embodiments, the stabilizing agents may beof a formula such as RXR′, XRX′ and RXR′X′. In the present invention,when the stabilizing agent is of the formula RXR′, XRX′ and RXR′X′, thenR and R′, or X and X′, can be the same subject molecule or molecularmoiety, or optionally they can be different subject molecules ormolecular moieties.

In some embodiments, R is selected from the group consisting of straightor branched siloxane, straight or branched carbosilane, straight orbranched alkyl, cycloalkyl, heterocyclyl, arylalkyl, heteroaryl, andcombinations thereof. In some embodiments, R is an alkyl, aryl, orsilicon-based polymer optionally substituted with one or more hydroxy,keto, carbonyl, carboxy, or combinations thereof. In some embodiments, Ris an ether, an ester, or combination thereof. In some embodiments, Rcomprises polyoxypropylene or polyoxyethylene.

In some embodiments, R is an alkyl, aryl, or silicon-based polymeroptionally substituted with one or more nitrogen-containing moietycomprising amine, amino, imine, imino, amide, ammonium, or combinationthereof.

In some embodiments, R can be alkyl, alkylene, alkyne, alkoxyalkyl,alkoxyalkoxyalkyl, alkylaminoalkyl, alkylaminooxyalkyl,alkoxyaminoalkyl, phosphonoalkyl, carboxyalkyl, carboxyalkoxyalkyl,wherein any one of which is optionally substituted with one or morehydroxy, carbonyl, halogen, phosphate, acetyl, ammonium, or combinationsthereof.

In some embodiments, R as defined herein can be optionally substitutedwith hydroxy, carboxy, amine, amine oxide, phosphate, acetate,carboxylate, ammonium derivatives of alkyl or aryl, or combinationsthereof. In some embodiments, R is optionally substituted with one ormore hydroxy, oxo, acetate, ammonium, carboxy, amino, or amine oxide.

In some embodiments, R is alkyl or aryl, wherein any one of which isoptionally substituted with one or more hydroxy, carbonyl, halogen,phosphate, acetyl, ammonium, or combinations thereof. Embodiments of theinvention include wherein R is alkyl, and alkyl is a C₁₋₃₀ alkyl, C₁₋₂₂alkyl, C₁₋₁₆ alkyl, C₁₋₁₂ alkyl, C₁₋₁₀ alkyl, or C₁₋₈ alkyl. In someembodiments, the alkyl moiety is C₄₋₃₀ alkyl, C₄₋₂₂ alkyl, C₄₋₁₈ alkyl,C₄₋₁₆ alkyl, C₄₋₁₄ alkyl, or C₄₋₁₂ alkyl. In some embodiments, the alkylmoiety is C₆₋₃₀ alkyl, C₆₋₂₂ alkyl, C₆₋₁₈ alkyl, C₆₋₁₆ alkyl, C₆₋₁₄alkyl, or C₆₋₁₂ alkyl. In some embodiments, the alkyl is a C₇₋₃₀ alkyl,C₇₋₂₂ alkyl, C₇₋₁₈ alkyl, C₇₋₁₄ alkyl, C₇₋₁₂ alkyl, or C₇₋₉ alkyl,optionally substituted with one or more hydroxy, carbonyl, halogen,phosphate, acetyl, ammonium, or combinations thereof.

Alkyl substituents can include ethyl, propyl, isopropyl, butyl,isobutyl, pentyl, hexyl, heptyl, octyl, 2-methyl butyl, 2-ethyl butyl,2-propyl butyl, 3-methyl butyl, 3-ethyl butyl, 3-propyl pentyl, 2-methylpentyl, 2-ethyl pentyl, 2-propyl pentyl, 3-methyl pentyl, 3-ethylpentyl, 3-propyl pentyl, 4-methyl pentyl, 4-ethyl pentyl, 4-propylpentyl, 2-methyl hexyl, 2-ethyl hexyl, 2-propyl hexyl, 3-methyl hexyl,3-ethyl hexyl, 3-propyl hexyl, 4-methyl hexyl, 4-ethyl hexyl, 4-propylhexyl, 5-methyl hexyl, 5-ethyl hexyl, 5-propyl hexyl, 2-methyl heptyl,2-ethyl heptyl, 2-propyl heptyl, 3-methyl heptyl, 3-ethyl heptyl,3-propyl heptyl, 4-methyl heptyl, 4-ethyl heptyl, 4-propyl heptyl,5-methyl heptyl, 5-ethyl heptyl, 5-propyl heptyl, 6-methyl heptyl,6-ethyl heptyl, 6-propyl heptyl, 2-methyl octyl, 2-ethyl octyl, 2-propyloctyl, 3-methyl octyl, 3-ethyl octyl, 3-propyl octyl, 4-methyl octyl,4-ethyl octyl, 4-propyl octyl, 5-methyl octyl, 5-ethyl octyl, 5-propyloctyl, 6-methyl octyl, 6-ethyl octyl, and 6-propyl octyl, any of whichcan be optionally substituted with one or more hydroxy, carbonyl,halogen, phosphate, acetyl, ammonium, or combinations thereof.

In some embodiments, R can be an olefin homolog of alkyl, with one ormore unsaturated carbon to carbon bond(s). That is, in some embodiments,any one of the substituents of R as described herein can be unsaturated.Examples of unsaturated substituents for R include, but are not limitedto, alkenyl, alkenoxy, alkenyloxyalkyl, alkenyloxyalkoxyalkyl,alkenylaminoalkyl, alkenylaminooxyalkyl, alkenyloxyaminoalkyl,phosphonoalkenyl, carboxyalkenyl, carboxyalkenyloxyalkyl,carboxyalkenylaminoalkyl, carboxyalkenylamidoalkyl. The term “alkenyl”refers to C₂₋₂₂ alkenyl groups, preferably C₄₋₁₈ alkenyl, or morepreferably C₆₋₁₈ alkenyl or C₇₋₁₄ alkenyl. The term alkenyl includes allstereoisomers, i.e., cis and trans isomers, as well as the E and Zisomers. In some embodiments, the unsaturated alkyl can comprise analkyne. The term “alkyne” refers to C₂₋₂₂ alkyne groups, preferablyC₄₋₁₈ alkyne, or more preferably C₆₋₁₈ alkyne or C₇₋₁₄ alkyne, whereinone or more triple bonds can exist in the alkyl chain.

In some embodiments, any one of the substituents as defined herein for Rcan be saturated.

In some embodiments, R can be a cyclic, polycyclic, or heterocyclicderivative of alkyl or aryl, i.e., R is a cycloalkyl, heterocyclyl,aryl, or heteroaryl. In some embodiments, the cycloalkyl, heterocyclyl,aryl or heteroaryl can be optionally substituted with one or morehydroxy, carbonyl, halogen, phosphate, acetyl, ammonium, or combinationsthereof. In some embodiments, the term cycloalkyl, heterocyclyl, aryl orheteroaryl can refer to a bicyclic ring, or a tricyclic ring.

The term “cycloalkyl” refers to a cyclized alkyl group that is saturatedor partially unsaturated. Cycloalkyl groups can include C₃₋₈ cycloalkyl.Typical cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl and cycloheptyl.

The term “heterocyclyl” or “heterocyclic” refers to a derivative ofcycloalkyl interrupted with Si, O, N, P, or combinations thereof. Morespecifically, the term “heterocyclyl” or “heterocyclic” is used hereinto refer to a saturated or partially unsaturated 3-7 memberedmonocyclic, or 3-14 membered bicyclic, ring system that consists ofcarbon atoms and from one to four heteroatoms independently selectedfrom the group consisting of Si, O, N, P, or combinations thereof.Examples include, but are not limited to, tetrahydrofuranyl,tetrahydropyranyl, pyrrolidinyl, piperidinyl, piperazinyl,pyrazolidinyl, dihydrofuranyl, morpholinyl, dihydroimidazolyl,dihydropyranyl, dihydrooxazolyl, tetrahydrooxazolyl,2-azabicyclo[2.2.1]heptanyl, 2,5-diazabicyclo[2.2.1]heptanyl, oxazinyl,isoxazinyl, oxathiazinyl, and the like. Heterocyclic groups can beoptionally substituted with one or more methyl, ethyl, oxo, halo,hydroxy, amino, alkylamino, hydroxymethyl, hydroxyethyl, hydroxypropyl,methoxymethyl, carboxy, or combinations thereof. In some embodiments,the term “heterocyclyl” refers to a cycloalkyl group that containsoxygen in the ring, i.e., a cyclic ether such as tetrahydrofuran ortetrahydropyran. In some embodiments, the term “heterocyclyl” refers toa cycloalkyl group that contains nitrogen and oxygen in the ring.

The term “aryl” refers to any aromatic carbon ring structure, or anycarbon ring structure with aromatic properties. Preferred aryls includeC₆₋₁₄ aryl, especially C₆₋₁₀ aryl, such as phenyl or naphthyl, and mostpreferably six carbon aryl. Aryl groups are optionally substituted withone or more methyl, ethyl, hydroxy, alkoxy, amino, alkylamino, halo,hydroxymethyl, hydroxyethyl, hydroxypropyl, methoxymethyl, or carboxy.Preferably aryl groups are optionally substituted with one or moremethyl, ethyl, halo, hydroxymethyl, hydroxyethyl, or carboxy.

The term “heteroaryl” refers to a derivative of aryl, wherein the arylring is interrupted with one or more Si, O, N, P, or combinationsthereof. More specifically, the term “heteroaryl” refers to 5-14membered heteroaromatic ring systems and most preferably to five or sixmembered heteroaromatic ring systems, wherein from one to four atoms inthe ring structure are heteroatoms independently selected from the groupconsisting of Si, O, N, P, or combinations thereof. Examples include,but are not limited to, tetrazolyl, pyridinyl, imidazolyl, isoxazolyl,furanyl, oxazolyl, thiazolyl, pyrrolyl, thienyl, pyrazolyl, triazolyl,e.g., 1,2,3-triazolyl and 1,2,4-triazolyl, isothiazolyl, oxadiazolyl,e.g., 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, and1,3,4-oxadiazolyl, oxatriazolyl, pyridazinyl, pyrimidinyl, pyrazinyl,triazinyl, e.g., 1,2,3-triazinyl and 1,2,4-triazolyl, quinolinyl,isoquinolinyl, indolyl, benzofuranyl, benzothienyl, benzimidazolyl, andindazolyl. In some embodiments, the term “heteroaryl” refers to an arylgroup that contains oxygen, nitrogen, or both in the ring.

In some embodiments, R is a fatty acid, or a homolog of a fatty acid.The term fatty acid refers to a saturated or unsaturated carboxyalkyl,carboxyalkoxyalkyl, carboxyalkylaminoalkyl, carboxyalkylamidoalkyl, orsalt or ester thereof, optionally substituted with hydroxy, C₁₋₄ alkyl,halo, or alkoxy. In some embodiments, the carboxyalkyl is a saturated orunsaturated C₂₋₃₀ carboxyalkyl, C₂₋₂₀ carboxyalkyl, C₂₋₁₈ carboxyalkyl,C₂₋₁₆ carboxyalkyl, C₂₋₁₄ carboxyalkyl, C₂₋₁₀ carboxyalkyl, or C₂₋₈carboxyalkyl, optionally interrupted with one or more Si, O, N, P, orcombinations thereof. In some embodiments, the term carboxyalkyl is aC₆₋₃₀ carboxyalkyl, C₆₋₂₀ carboxyalkyl, C₆₋₁₈ carboxyalkyl, C₆₋₁₆carboxyalkyl, C₆₋₁₄ carboxyalkyl, C₆₋₁₀ carboxyalkyl, or C₆₋₈carboxyalkyl, optionally substituted with one or more Si, O, N, P, orcombinations thereof. In some embodiments, the carboxyalkyl is butanoicacid, hexanoic acid, octanoic acid, decanoic acid, dodecanoic acid,tetradecanoic acid, hexadecanoic acid, octadecanoic acid, icosanoicacid, or docosanoic acid.

In some embodiments, R is an ester of a fatty acid, i.e., R is acarboxyalkyl optionally interrupted with an ester. Examples ofcarboxyalkyls interrupted with an ester include, but are not limited to,carboxyalkoxyalkyl or carboxyalkoxyalkoxyalkyl. In some embodiments, Ris an alkanoamide derivative of a fatty acid, i.e., R is a carboxyalkylinterrupted with an amido group. For example, in some embodiments R is acarboxyalkylamido or carboxyalkylamidoalkyl. In some embodiments, R isan oxyethylated fatty acid.

In the present invention, R can be a derivative of fatty acid optionallyinterrupted with Si, O, N, P, or combinations thereof, i.e., in someembodiments, R is a carboxyalkyl optionally interrupted with Si, O, N,P, or combinations thereof. In some embodiments, R is a carboxyalkyloptionally substituted with a halogen. In some embodiments, R is acharged carboxyalkyl, i.e., R is an anionic or cationic derivative of afatty acid.

In some embodiments, R is an alcohol or alcohol derivative. For example,R can be an alkylphenol, or an ester of an alcohol. In some embodiments,R is an alcohol or phenol optionally interrupted with Si, O, N, P, orcombinations thereof, optionally substituted with a halogen, or both.

In some embodiments, R is an ester or polyester derivative of an alkyl.For example, in some embodiments, the R is a saturated or unsaturatedalkoxyalkyl, alkoxyalkoxyalkyl, or alkoxyalkoxyalkoxyalkyl, preferablyC₂₋₂₀ alkoxyalkyl, C₂₋₂₀ alkoxyalkoxyalkyl, or C₂₋₂₀alkoxyalkoxyalkoxyalkyl; a C₅₋₁₆ alkyl, C₅₋₁₆ alkoxyalkyl, C₅₋₁₆alkoxyalkoxyalkyl, C₅₋₁₆ alkoxyalkoxyalkoxyalkyl; or a C₇₋₁₄alkoxyalkyl, C₇₋₁₄ alkoxyalkoxyalkyl, or C₇₋₁₄ alkoxyalkoxyalkoxyalkyl;any one of which can be optionally substituted with one or more hydroxy.In some embodiments, the term polyester is a compound of the generalformula [(CH_(q))_(n)CO₂X_(p)]_(m), wherein X is C₁₋₁₀ alkyl, C₁₋₁₀alkenyl, or C₁₋₁₀ alkoxyalkyl, p is 0 or 1, q is 1 or 2, n is 1-10, andm is 1-100, preferably 1-20 or 1-10. In some embodiments, the ester orpolyester derivative of alkyl is optionally substituted with one or morehydroxy.

In some embodiments, R is a polyoxyalkylene. The term polyoxyalkylenerefers to an alkylene subject molecule or molecular moiety interruptedwith two or more oxygen atoms. In some embodiments, the termpolyoxyalkylene refers to a C₁₋₂₁, O₁₋₁₄ polyoxyalkylene, or a C₁₋₁₄,O₁₋₇ polyoxyalkylene. Examples of polyoxyalkylenes suitable for use inthe present invention include, but are not limited to, polyoxyethylene,polyoxypropylene, polyoxybutylene, polyoxypentylene, polyoxyhexylene,polyoxyheptylene, or polyoxyoctylene. In some embodiments, an R is acopolymer of two or more polyoxyethylene, polyoxypropylene,polyoxybutylene, polyoxypentylene, polyoxyhexylene, polyoxyheptylene, orpolyoxyoctylene groups. In some embodiments, R is an oxyethylatedcopolymer with other organic or inorganic moieties.

In some embodiments, R can be a polyamine, amine oxides, amineethoxylates and other amine derivatives of alkyl. The term “polyaminederivative” refers to an alkyl chain wherein two or more carbon atoms inthe alkyl chain are replaced with nitrogen. The term also includesmonoalkyl amines as well as dialkyl amines. The term “amine oxidederivative” refers to an alkyl chain wherein two or more carbon atoms inthe alkyl chain have been replaced with a nitrogen and an oxygen (i.e.,—N—O—). For example, R can be alkylaminooxyalkyl,carboxyalkylaminooxyalkyl, alkylaminooxyalkyl, or alkoxyaminoalkyl. Insome embodiments, the polyamine derivative or amine oxide derivative isoptionally substituted with an ethyoxylate.

In some embodiments, R is a silicon-based polymer. The term“silicon-based polymer” refers to linked monomers, each monomercontaining at least one silicon atom. For example, the termsilicon-based polymer can include, but is not limited to, siloxanes,silicones, or carbosilanes. In some embodiments, the silicon-basedpolymer can be end-blocked with a non-silicon containing end group.

In some embodiments, R is a straight, branched, or cyclic siloxane. Insome embodiments, R is a siloxane optionally substituted with one ormore hydroxy, carbonyl, alkyl, halogen, haloalkyl, hydoxyalkyl,phosphate, acetyl, ammonium, or combinations thereof. In someembodiments, R is a Si₁₋₂₁, O₁₋₂₁ siloxane, a Si₃₋₁₄, O₃₋₁₄ siloxane, ora Si₅₋₁₀, O₅₋₁₀ siloxane. In other embodiments, R is a carbosilane. Theterm carbosilane refers to a polymer with carbon and silicon in itsbackbone. In some embodiments, R is a carbosilane optionally substitutedwith one or more hydroxy, carbonyl, alkyl, halogen, haloalkyl,hydroxyalkyl, phosphate, acetyl, ammonium, or combinations thereof. Insome embodiments, R is a Si₁₋₂₁, C₁₋₂₁ carbosilane, a Si₃₋₁₄, C₁₋₁₄carbosilane, or a Si₅₋₁₀, C₅₋₁₀ carbosilane. In some embodiments, thesiloxane or carbosilane is end-blocked, e.g., end-blocked with vinyl orsilanone. In some embodiments, R is a siloxane or carbosilane optionallysubstituted with one or more alkyl, halogen, haloalkyl, hydoxyalkyl, orcombinations thereof. For example, in some embodiments, the siloxane orcarbosilane can be methyl, ethyl, propyl, butyl, or pentyl substituted,or combinations thereof.

In some embodiments, R comprises copolymers of silicone with polyether,polyester, polyol, polyamine, polyurethane, polyoxyethylene,polyoxypropylene, and other polymeric moieties. For example, in someembodiments, R is a siloxane or carbosilane optionally substituted withone or more polyester, polyol, polyamine, polyurethane, polyoxyethylene,polyoxypropylene, or combination thereof. The siloxane or carbosilane ofthe present invention can include their salts, ethers, and esters. Insome embodiments, R is a charged siloxane or carbosilane, e.g., acationic derivative or anionic derivative of a siloxane. In someembodiments, R is a salt of a siloxane or carbosilane, e.g., an ammoniumsalt. In some embodiments, R comprises silicones derivatized with longchain ester, long chain alcohol, long chain amine, or combinationsthereof.

Various X functional groups can be provided by the present invention. Insome embodiments, X is an oxoanion or oxide of sulfur or phosphorus, anoxoanion or carbonyl derivative of nitrogen, a carboxylate, a borate, ora silicate. In some embodiments, X is sulfate, sulfonic acid, sulfonate,sulfite, sulfoxide, taurate, sulfosuccinate, sulfobetaine,sulfatobetaine, sulfonamide, phosphate, phospho, phosphono, phosphinyl,nitrate, nitrite, amino, imino, isocyano, isothiocyano, acetamido,acetimido, azido, diazo, cyano, cyanato, alkylcarboxylate, acrylate,sebacate, phthalate, borate, tetraborate, orthosilicate, metasilicate,metal silicate, chlorite, chloride, iodide, iodate, bromide, bromate,fluoride, or fluorate. In some embodiments, X is sulfate, sulfonic acid,sulfonate, sulfosuccinate, or taurate.

In some embodiments, X is a functional group that contains a sulfuratom. Examples include, but are not limited to any functional group thatcontains sulfate, sulfonate or sulfonic acid. The term “oxoanion oroxide of sufur” refers to a sulfate, sulfonic acid, sulfonate, sulfite,taurate, sulfosuccinate, sulfobetaine, sulfonamide,methoxymethanilamide, sulfamyl, or sulfeno.

In some embodiments of the present invention, X can be any functionalgroup that contains a halogen. Examples include, but are not limited to,any functional group that contains a chloro, flouro, bromo, iodo group,or combinations thereof. In some embodiments, X can be chlorite,chloride, iodide, iodate, bromide, bromate, fluoride or fluorate.

In some embodiments of the present invention, X can be any functionalgroup that contains a nitrogen. Examples include, but are not limitedto, any functional group that contains a nitrate, nitrite, nitroamine,amino, imino, isocyanoto, isothiocyano, acetamido, acetimido, azido,diazo, cyano, cyanato, nitroso, nitrosoimino, nitramino, nitro, orcombinations thereof.

In some embodiments, X can be any functional group that contains aphosphorus. The term “oxoanion or oxide of phosphorous” refers to aphosphate, phospho, phosphono, phosphinyl, or phosphino.

In some embodiments, X is carboxylate, acrylate, sebacate, phthalate,acetate, or oxide. In some embodiments, X can be any functional groupthat contains a boron. Examples include, but are not limited to borate,peroxoborate, tetraborate, or boranate.

In the present invention, X can be any functional group that contains asilicon atom. Examples include, but are not limited to, silane,orthosilcate, metasilicate, or a metal silicate.

Many stabilizing agents were screened to determine which, if any, couldreplace the alkyl amine fluoro-surfactant and yet have no negativeimpact on the accuracy of the assays. Without being limited by theory,we postulate that the stabilizing agents maintain a sample's ratiobetween free analyte and bound analyte thereby preventing the disruptionof the free unbound analyte versus bound analyte equilibrium found inthe biological fluid of a subject or in an in vitro system.

In one embodiment, the stabilizing agent comprises the general formulaRX, wherein R comprises a chemical group containing an alkyl, an aryl, afatty acid, an alcohol, an ester, an ether, an oxyalkylate, or asiloxane, and X comprises a chemical group containing sulfur. When RXcomprises a halogen, the halogen moiety may be any halogen, e.g., F, Cl,Br, or I, with the proviso that the present invention does not comprisean alkyl amine fluoro-surfactant, such as FC100.

In one embodiment, the present invention identifies the stabilizingagent as an alkyl alcohol derivative compound, for example 2-ethyl-hexylsulfate (EHS). The general chemical structure of such alkyl alcoholderivative compounds is C_(n)H_(2n+1)X wherein n is about 6 to about 18and X is a sulfate derivative, for example SO₄ ⁻Na⁺ for 2-ethyl-hexylsulfate sodium salt having the chemical structure:CH₃(CH₂)₃CH(C₂H₅)CH₂OSO₃ ⁻Na⁺. X may also be any of thesulfur-containing groups disclosed in FIG. 2, Group X.

Other stabilizing agents suitable for use in the present inventioninclude: 1-hexane sulfonic acid, or salt thereof (for example, Na+salt); 1-heptane sulfonic acid, or salt thereof (for example, Na+ salt);1-octane sulfonic acid, or salt thereof (for example, Na+ salt);1-decane sulfonic acid, or salt thereof (for example, Na+ salt); sodiumC₁₄₋₁₆ olefin sulfonate (CAS# 68439-57-6); sodium dodecyl sulfate;sodium dioctyl sulfosuccinate; sodium N-oleyl-N-methyltaurate; sodiumpolyoxyethylene lauryl sulfate (molecular weight: 346); aminealkylbenzyl sulfonate (molecular weight: 385); and sodium ethyl-hexylsulfate.

The present invention provides new and useful compositions for use inthe diagnostic measurement of free or unbound analyte concentrations ina fluid. In some embodiments, the analyte is present in a biologicalfluid. The biological fluid may be plasma or serum. It may also becomprised of seminal fluid, saliva, urine, fecal solutions, cerebralspinal fluid, or gastric fluids. Generally, however, the biologicalfluid is plasma or serum. The compositions of the present invention mayalso be used in an in vitro system comprising cell culture supernate orfiltrate. The term “biological fluid” includes fluids from a biologicalorganism, as well as fluids derived from a biological organism, e.g.,fluids that have been fractionated, diluted, chemically modified, orcombinations thereof.

The analyte may be any small molecule such as, but not limited to, ahormone, a drug, or a vitamin. In some embodiments, the analyte ispresent in an equilibrium state between the free unbound condition andthe bound condition (e.g. protein bound). For example, the analyte canbe a thyroid hormone or vitamin B12. Other analytes suitable for use inthe present invention include the following classes of vertebratehormones:

Amine Hormones:

-   -   a. Tyrosine-derived (Simple aromatic rings)        -   Catecholamines (e.g. adrenaline, noradrenaline, dopamine).        -   Thyroid hormones (e.g. T4, or isomers of T3, T2, and T1).    -   b. Tryptophan-derived (Polycyclic and heterocyclic aromatic        compounds)        -   Melatonin, serotonin.

Peptide Hormones:

-   -   a. Small peptides, e.g.        -   Angiotensin IV has 6 amino acid residues, Angiotensin III &            Angiotensin II have 7 & 8 amino acids, respectively. Due to            the small molecular size, these peptides are likely bound to            plasma carrier molecules, such as proteins.        -   Brain natriuretic peptide (BNP), 32 amino acids. Other            natriuretic peptide hormones include atrial natriuretic            peptide (ANP) and C-type natriuretic peptide (CNP).        -   Calcitonin, 32 amino acids peptide, derived from the            precursor, procalcitonin.        -   Adrenocorticotropic hormone (ACTH) has 39 amino acids.        -   Insulin is a 51 amino acids peptide.        -   Parathyroid hormone (PTH), 84 amino acids. PTH can also            occur in several different fragments having different            clinical utilities: intact PTH; N-terminal PTH; mid-molecule            PTH, and C-terminal PTH.    -   b. Large peptides (large peptides yield smaller protein        molecules after dissociation of protein multimers or after        fragmentation. These smaller molecules may circulate in an        equilibrium with carrier molecules), e.g.:        -   Follicle-stimulating hormone (FSH).        -   Luteinizing hormone (LH).        -   Thyroid-stimulating hormone (TSH).        -   Chorionic gonadotropin (e.g. hCG).        -   Thyrotropin-releasing hormone (TRH).        -   Prolactin (PRL).        -   Erythropoietin (EPO).

Steroid or Sterol Hormones: This invention is useful for those steroidor sterol hormones that have a free form in equilibrium with a form thatis bound to plasma transport molecules, such as proteins. The free formis biologically active and has clinical utility.

-   -   a. Steroid hormones, e.g:        -   Cortisol: About 4% of circulating cortisol is free and            therefore available to receptors. The remainder is bound to            proteins including corticosteroid binding globulin (CBG) and            albumin.        -   Testosterone: About 2-3% of the circulating testosterone is            free; the remainder is bound to testosterone binding            proteins including sex hormone binding globulin (SHBG, about            44%) and cortisol binding globulin (CBG, about 3.5%), as            well as to albumin (about 50%).        -   Dehydroepiandrosterone (DHEA). A small percentage of DHEA            occurs as a free form, although the majority is strongly            bound to sex steroid binding globulin and weakly bound to            corticosteroid binding globulin and albumin.        -   Progesterone: A small percentage of progesterone occurs as a            free form, although the majority is bound to cortisol            binding globulin and albumin.        -   Estriol: Estriol occurs as a free form or bound to sex            hormone binding globulins.        -   Estradiol: About 1-3% of the circulating hormone is free            while the remainder is strongly bound to estrogen binding            globulin.    -   b. Sterol hormones:        -   Vitamin D derivatives.        -   Calcitriol.

The term thyroid hormone refers to thyroxine (T4), triiodothyronine(T3), diiodothyronine (T2), monoiodothyronine (T1), and combinationsthereof. It is understood that the thyroid hormones can be comprised oftheir salts, their L- or D-enantiomers, or their de-iodinated andisomeric forms.

For purposes of the present invention, it is understood that the term“analyte” encompasses all enantiomers and isomers of that particularanalyte, either in a mixture or a homogenous environment. For analytesin a biological fluid, the analyte can originate either exogenously orendogenously. In some embodiments, the analyte can be in a biologicalfluid from a primate such as apes, monkeys, orangutans, baboons,gibbons, and chimpanzees; canids such as dogs and wolves; felids such ascats, lions, and tigers; equids such as horses, donkeys, and zebras,food animals such as cows, pigs, and sheep; and rabbits, mice, rats,guinea pigs, and ferrets. The analyte can also be in a fluid from amodel animal, e.g., disease model animal such as mice, rats, or otherlaboratory animal; an economically valuable animal, e.g., economicallyimportant breeding stock, racing animals, show animals, heirloomanimals, rare or endangered animals, or companion animals. Inparticular, the analyte is in a biological fluid from a human. The term“subject,” as used herein, refers to any mammal, including humans andnon-humans, such as, but not limited to, domestic and farm animals, zooanimals, sports animals, and pets.

The stabilizing agent may be incorporated into a reagent buffer, or awash buffer, or both. It can also be mixed with a capture ligand. Underother embodiments, it can be a separate solution.

In one embodiment, the present invention is directed to a compositionfor use in an assay measuring the concentration of an analyte, in someembodiments a free analyte, the composition comprising a capture ligandand a stabilizing agent.

Capture ligands are analyte-specific and may comprise any substancecapable of selectively binding the free analyte of interest. Examples ofcapture ligands include, but are not limited to, antibody, antibodyfragment, antibody mimic, or analyte-specific binding protein such asintrinsic factor or folate-binding protein, and combinations thereof.

As used herein, the term “antibody” is intended to include all formssuch as, but not limited to, polyclonal, monoclonal, purified IgG,purified IgM, purified IgA, or combinations thereof; single chainantibodies (U.S. Pat. No. 4,946,778), chimeric or humanized antibodies(Morrison et al., 1984, Proc. Nat'l Acad. Sci. USA 81:6851; Neuberger etal., 1984, Nature 81:6851) and complementary determining regions (CDR;see Verhoeyen and Windust, in Molecular Immunology 2ed., by B. D. Hamesand D. M. Glover, IRL Press, Oxoford University Press, 1996, at pp.28-325). The term “antibody fragment” includes, but is not limited to,fragments such as Fv, single chain Fv (scFv), F(ab′)₂, and Fab fragments(Harlow and Leon, 1988, Antibody, Cold Spring Harbor). Antibodies andantibody fragments of the present invention can be obtained by anyconventional methods, such as, but not limited to, the methods describedin Antibodies: A Laboratory Manual (E. Harlow, D. Lane, Cold SpringHarbor Laboratory Press, 1989) that is incorporated herein by referencein its entirety.

The term “antibody mimic” refers to chemicals that mimic the functionsof antibodies. Antibody mimics are generally small in size, allowingthem to avoid provoking an immunogenic response. There are severalapproaches to the structure and manufacture of these antibody mimics,such as alternative protein frameworks; structures comprising RNA;unnatural oligomers such as benzodiazepines, beta-turn mimics, proteaseinhibitors and purine derivatives; unnatural biopolymers such asoligocarbamates, oligoureas and oligosulfones; and the joining ofvarious substituents to scaffolds such as xanthene and cubane asdescribed in Hsieh-Wilson, et al., (1996) Acc. Chem Res. 29:164-170, andU.S. Pat. No. 5,770,380.

The term “intrinsic factor” refers to a glycoprotein necessary for theabsorption of vitamin B₁₂. In some embodiments, the term “instrinsicfactor” refers to human intrinsic factor.

The term “analyte-specific binding protein” can include any non-antibodyprotein that binds specifically to an analyte. Examples of analytes withtheir analyte-specific binding proteins include, but are not limited to:thyroxine/thyroxine-binding globulin; enzyme inhibitor, coenzyme orcofactor/enzyme; cortisol/cortisol binding protein; vitaminB12/intrinsic factor; and folate/folate-binding protein. In someembodiments, the analyte-specific binding protein is the capture ligand.In some embodiments, the analyte-specific binding protein isfolate-binding protein or intrinsic factor.

In one embodiment, the composition of the present invention can comprisea capture ligand, a stabilizing agent, and can further comprise salts,such as ammonium salts, sodium salts, potassium salts, calcium salts,magnesium salts, zinc salts, chloride salts, carboxylate salts,phosphate salts, inorganic sulfate salts, or combinations thereof.

In one embodiment, the present invention is directed to a compositionfor use in an assay measuring the concentration of an analyte. In someembodiments, the analyte exists as a free analyte and a bound analyte,and the invention is directed to an assay measuring the concentration ofthe free analyte. The term “free analyte” refers to an analyte that isnot bound, either specifically or nonspecifically, to other molecularspecies such as proteins, other than a capture ligand. Thus, the freeanalyte is not associated with other molecular species in the fluid. Theterm “bound analyte” refers to an analyte that is associated, eitherspecifically or nonspecifically, with other molecular species such asproteins, other than a capture ligand. In some embodiments, to stabilizethe ratio of free to bound analyte, the present invention employs one ormore stabilizing agents comprising the general formula RX.

The stabilizing agent can be present in various concentrations. Whendescribing the concentration of the stabilizing agent herein, the term“volume percent” refers to the volume of the stabilizing agent per unitvolume of the final composition. The term “final composition” refers tothe composition comprising the capture ligand and the stabilizing agent.Thus, e.g., if 1 ml of stabilizing agent was added to 49 ml of washbuffer, and the stabilizing agent/wash buffer was placed in 50 ml of asolution containing the analyte and the capture ligand, then the volumepercent would be 1% of stabilizing agent (i.e., 1 ml stabilizing agentin 100 ml final composition). Suitable volume percents of a stabilizingagent in a final composition include, but are not limited to, about0.00001 to about 0.5 volume percent, about 0.00005 to about 0.4 volumepercent, about 0.00009 to about 0.3 volume percent, about 0.0001 toabout 0.1 volume percent, about 0.0002 to about 0.2 volume percent, orabout 0.00015 to about 0.15 volume percent, wherein the above volumepercents are based on the total volume of a final composition. Since thebinding affinity of an analyte for its analyte-specific binding proteindetermines the equilibrium between free analyte and bound analyte forms,one of skill in the art can optimize the percentage of stabilizing agentfor a particular analyte on a case by case basis.

The concentration of the stabilizing agent can also be measured in molesof stabilizing agent per liter of the final composition. Theconcentration of a stabilizing agent in a final composition can begenerally about 1 to about 900 micromolar, about 5 to about 850micromolar, about 25 to about 800 micromolar, about 40 to about 800micromolar, about 5 to about 750 micromolar, about 30 to about 700micromolar, or about 25 to about 600 micromolar.

In embodiments wherein the stabilizing agent is EHS, the volume fractionof EHS in a final composition is generally about 0.0001 to about 0.05volume percent, about 0.0005 to about 0.04, about 0.0009 to about 0.03,about 0.001 to about 0.02, about 0.002 to about 0.019, about 0.004 toabout 0.015, or about 0.005 to about 0.01 volume percent, wherein theabove volume percents are based on the total volume of a finalcomposition.

In embodiments wherein the stabilizing agent is EHS, the concentrationof EHS in a final composition can be generally about 50 to about 800micromolar, about 100 to about 750 micromolar, about 150 to about 700micromolar, about 150 to about 650 micromolar, about 200 to about 600micromolar, about 250 to about 550 micromolar, or about 300 to about 500micromolar.

With regard to the methods of the present invention, it is understoodthat the stabilizing agent can be present either upon mixing of thesample with the analyte-specific capture ligand or immediatelythereafter. For example, the stabilizing agent can be added sequentiallyfollowing the combination of the sample with the analyte-specificcapture ligand as a component of a reagent buffer, a wash buffer, orboth.

In certain embodiments, the present invention is also directed to amethod for measuring a concentration of a free analyte, the methodcomprising:

-   -   (a) adding a capture ligand for the free analyte to a vessel;    -   (b) adding a stabilizing agent to the vessel;    -   (c) adding a sample comprising the free analyte to the vessel;    -   (d) adding a detection system to the vessel; and    -   (e) measuring the concentration of the free analyte in the        sample using the detection system.

It is recognized that the steps of the method for measuring aconcentration of a free analyte can be performed in various sequentialorders, with the proviso that step (e) must be the final step. Thus,e.g., step (b) can precede step (a), step (c) can precede step (b), orboth,

In some embodiments, the vessel may comprise a vial, a tube, a containerfor a reaction mixture, a microtiter plate well, a membrane in a lateralflow system, or other stable components of a liquid flow system.

The term “detection system” as used herein refers to one or morecomponents of a system known to detect, either directly or indirectly,an analyte.

In some embodiments, the detection system comprises components ofradioimmunoassays, enzyme-linked immunosorbent assays, enzymeimmunoassays, chemiluminescent immunoassays, bioluminescentimmunoassays, or fluorescent immunoassays. Such detection systems arewell known in the literature. In some embodiments, the detection systemcomprises paramagnetic particles coated with a capture ligand-specificbinding molecule, detectable label, or combination thereof. In someembodiments, the capture ligand-specific binding molecule ishapten-specific, and the capture ligand is haptenated. For example, insome embodiments the hapten is biotin. In some embodiments, the captureligand-specific binding molecule is selected from the group consistingof streptavidin, avidin, biotin-specific antibody, biotin-specificantibody fragment, and biotin-specific antibody mimic.

In some embodiments, the detection system comprises a detectable label.As used herein, a “detectable label” has the ordinary meaning in the artand refers to an atom (e.g., radionuclide), molecule (e.g.,fluorescein), or complex that is or can be used to detect (e.g., due toa physical or chemical property) or to indicate the presence of amolecule, or to reveal binding of another molecule to which it iscovalently bound or with which it is otherwise associated. The term“label” also refers to covalently bound or otherwise associatedmolecules (e.g., a biomolecule such as an enzyme) that act on asubstrate to produce a detectable atom, molecule or complex. Detectablelabels suitable for use in the present invention include any compositiondetectable by spectroscopic, photochemical, radiological, biochemical,immunochemical, electrical, optical or chemical means.

Labels useful in the present invention include fluorescent dyes (e.g.,fluorescein, Texas red, rhodamine, green fluorescent protein, enhancedgreen fluorescent protein, lissamine, phycoerythrin, Cy2, Cy3, Cy3.5,Cy5, Cy5.5, Cy7, Fluor X, SyBR Green I & II [Molecular Probes], and thelike), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g.,hydrolases, particularly phosphatases such as alkaline phosphatase,esterases and glycosidases, or oxidoreductases, particularly peroxidasessuch as horseradish peroxidase, and others commonly used in ELISAs),substrates, cofactors, inhibitors, chemiluminescent groups, chromogenicagents, and colorimetric labels such as colloidal gold or colored glassor plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.Techniques for detecting such labels are well known in the field.

Thus, for example, radiolabels may be detected using scintillationcounters, chemiluminescent labels and fluorescent markers may bedetected using a photodetector to detect emitted light (e.g., as influorescence-activated cell sorting). Enzymatic labels are typicallydetected by providing the enzyme with a substrate and detecting thereaction product produced by the action of the enzyme on the substrate,and colorimetric labels are detected by simply visualizing the coloredlabel.

Thus, a label is any composition detectable by spectroscopic,photochemical, radiological, biochemical, immunochemical, electrical,optical or chemical means. The label may be coupled directly orindirectly to the desired component of the assay according to methodswell known in the art. Non-radioactive labels are often attached byindirect means. In some embodiments, a detectable label that is eitherinherently detectable or covalently bound to a signal generating systemsuch as a detectable enzyme, fluorescent compound, or chemiluminescentcompound (e.g., T4-ALP or T3-ALP in a competitive immunoassay for FreeT4), competes with analyte (e.g., Free T4 in a fluid sample) for bindingto a capture ligand (e.g., T4-specific monoclonal antibody). In someembodiments, the capture ligand may be directly immobilized on a solidphase. In some embodiments, the capture ligand may be biotinylated forindirect immobilization on a solid phase through its binding with acapture ligand-specific binding molecule such as streptavidin orbiotin-specifc monoclonal antibody.

Where an analyte has a natural, analyte-specific binding molecule, suchan analyte can be detected or measured by using a labeled form either ofthe analyte or an analog of the analyte, or of the analyte-specificbinding molecule in a detection system. In some embodiments, a captureligand (e.g., Intrinsic Factor (IF)-specific monoclonal antibody)competes with analyte (e.g., vitamin B12) for binding to a natural,analyte-specific detectable label (e.g., IF-ALP in a competitiveimmunoassay for vitamin B12). In some embodiments, the capture ligandmay be directly immobilized on a solid phase. In some embodiments, thecapture ligand may be biotinylated for indirect immobilization on asolid phase through its binding with a capture ligand-specific bindingmolecule such as streptavidin or biotin-specifc monoclonal antibody. Thedetectable label is either inherently detectable or covalently bound toa signal generating system such as a detectable enzyme, fluorescentcompound, or chemiluminescent compound. Such detectable labels can beconjugated directly to signal generating compounds, e.g., by conjugationwith an enzyme or fluorophore. Techniques for detecting labels are wellknown in the field and include, but are not limited to, use of achemiluminescent agent, a colorimetric agent, an energy transfer agent,an enzyme, a substrate of an enzyme reaction, a fluorescent agent or aradioisotope. The enzyme may be selected from the group containingalkaline phosphatase, amylase, luciferase, catalase, beta-galactosidase,glucose oxidase, glucose-6-dehydrogenase, hexokinase, horseradishperoxidase, lactamase, urease and malate dehydrogenase. The substratecomprises those molecular groups upon which the preceding labels areknown to act.

In some embodiments of the present invention the capture ligand isimmobilized on a solid phase. A solid phase may be comprised of aprotein coupling surface including, for example, a microtiter plate, acolloidal metal particle, an iron oxide particle, a polymeric bead,nanoparticles or microparticles. Further, the solid phase may becomprised of chemical or molecular aggregates that function as a solidphase in a separation system (e.g. fractionation, precipitation, orcentrifugation).

In certain embodiments, the present invention is also directed to a kitfor use in estimating a concentration of a free analyte, the kitcomprising:

-   -   (a) a capture ligand for the free analyte;    -   (b) a stabilizing agent; and    -   (c) a detection system.

In some embodiments, the kit further comprises (d) a reference standard.Various reference standards can be used, depending on the analyte ofinterest. Generally, the reference standard comprises an analyte or itsanalog that has a known concentration and is used to estimate theconcentration of the particular analyte to be measured. The referencestandard can comprise a purified analyte, a recombinant, native orsynthetic analyte, a drug, a hormone or a vitamin. The matrix portion ofa reference standard matrix comprises a biological or synthetic fluidthat can approximate the environment of the analyte in the sample to bemeasured.

In one particular embodiment, the present invention improves theaccuracy of assays measuring concentrations of free non-protein boundthyroid hormones by using 2-ethyl-hexyl sulfate (EHS) and/or relatedcompounds as a stabilizing agent. The present invention further improvesdiagnostic accuracy of measuring the bioavailable or free portion ofthyroid hormones present in body fluid of a subject. The improvement inconcentration accuracy measurements occurs by stabilizing theequilibrium of the free non-protein bound and protein-bound hormoneinherent in a body fluid, such as serum or plasma.

In this embodiment, the present invention uses sulfonated or sulfatedsmall molecules as the anionic salt component of the stabilizing agent.Traditionally, sulfates or sulfonates such as 8-anilinonaphthalene-1-sulfonate (ANS) are used to displace protein boundanalytes in order to measure total (bound plus free) hormone. Therefore,the inclusion of molecules with sulfonate or sulfate groups into thecomposition as stabilizing agents is expected to result in an artificialincrease in the free-hormone fraction or an over-sampling diagnosticerror. The present invention, therefore, is unexpected.

In this embodiment, surfactants or non-surfactants with sulfonate orsulfate groups improve the consistency of immunoassays by being added tothe assay reaction as stabilizing agents. Without being limited bytheory, it is postulated that the mechanism for enabling accurateconcentration measurements of free thyroid hormones is by stabilizationof the free to bound hormone equilibrium through the addition of EHS, orother small chain (6 to 18 carbons) anionic sulfates or sulfonates,early in the reaction. Among the various sulfated or sulfonatedchemicals, those with sterically available sulfates or sulfonates act assuitable stabilizing agents. In some embodiments, other factorsimportant for suitable stabilizing agents are described by a generalformula RX, wherein R comprises a short chain hydrocarbon (that confersthe hydrophobicity to one end of the molecule) and wherein X comprises asmall hydrophilic group, e.g., a small chemical group containing morethan one oxygen atom. FIGS. 1 and 2 illustrate, without intending to belimiting, some of the various R groups and X groups that can be combinedto form a stabilizing agent of the present invention.

In one embodiment of the present invention, the analyte is a thyroidhormone. The following are points of interest to note regarding thyroidhormones.

-   -   1. Thyroxine (T4) is an essential hormone produced by the        thyroid gland. Triiodothyronine (T3) can be directly released        from thyroglobulin in the thyroid gland but most T3 is        manufactured in other parts of the body by deiodination of        thyroxine.    -   2. Via a reaction with the enzyme thyroperoxidase, iodine is        covalently bound to tyrosine residues in thyroglobulin        molecules, forming monoiodotyrosine (MIT) and diiodotyrosine        (DIT). Thyroxine is produced by linking two moieties of DIT.        Combining one molecule of MIT and one molecule of DIT produces        triiodothyronine. MIT and DIT are formed in situ on        thyroglobulin as inactive precursors of T4 and T3 hormones.        Proteases in lysosomes digest iodinated thyroglobulin, releasing        T3 and T4. MIT and DIT are also products of proteolysis, but        they are degraded in situ by iodotyrosine dehalogenase.    -   3. Other constituents of the thyroid gland besides T4 and T3        include diiodothyronine (T2) and monoiodothyronine (T1).    -   4. Thyronamines are decarboxylated and deiodinated metabolites        of the thyroid hormones thyroxine (T4) and        3,5,3′-triiodothyronine (T3).    -   5. The follicular cells in the thyroid gland synthesize the        iodine-containing thyroid hormones, T4 and T3; the        parafollicular cells of the thyroid gland produce calcitonin, a        32 amino acid peptide hormone cleaved from the larger        polypeptide procalcitonin.

Thyroxine (T4), triiodothyronine (T3), diiodothyronine (T2), andmonoiodothyronine (T1) in blood can be bound to serum proteins, andtherefore only a fraction may be distributed in the free non-proteinbound form, otherwise known as the bioavailable portion. This freeamount is either increased or decreased in a thyroid disease state of asubject. For example, in hypothyroidism the free non-protein boundfraction in blood is decreased, whereas in hyperthyroidism thenon-protein bound fraction is elevated.

The free thyroxine (FT4) test is used as a direct measurement of thyroidfunction and is commonly requested by physicians as a follow-up to, orin conjunction with, the thyroid stimulating hormone (TSH) test, inorder to determine whether the thyroid status of a subject is euthyroid(healthy thyroid function), hypothyroid or hyperthyroid. However, it ismore common to encounter misleading FT4 tests than misleading TSHmeasurements due to the binding-protein dependencies inherent with allfree non-protein bound thyroid hormones and other interference factorsthat can alter the free to bound equilibrium. Therefore, it is importantto include a stabilizing agent to stabilize FT4 when testing a samplefrom a subject for proper thyroid function.

In one embodiment of the present invention, the stabilizing agent isadded to the assay before the sample containing the analyte is added. Inother embodiments, the stabilizing agent can be added to the assay afterthe sample containing the analyte is added, e.g., as part of a reagentbuffer. In some embodiments, the stabilizing agent is a part of areagent kit containing a capture ligand (e.g., an analyte-specificantibody). In some embodiments, the stabilizing agent of the presentinvention can be added to an analyte-specific assay, e.g., FT4 assay, invarious ways. For example, the stabilizing agent can be added by meansof a vial system, a buffer system, or a pack system. In the vial system,the stabilizing agent, e.g., one or more sulfonated compounds, is addedfrom vials to the reaction mixture. In the buffer system, thestabilizing agent, e.g., one or more sulfonated compounds, is part of awash buffer solution which is added to the reaction mixture. In the packsystem, the stabilizing agent, e.g., one or more sulfonated compounds,is taken from the well of the reagent pack containing theanalyte-specific antibody and added to the vessel containing thereaction mixture.

BCI's Access FT4 assay system (BCI, Fullerton, Calif.) is used tomeasure the concentration of free non-protein bound thyroxine in theserum or plasma of subjects. This assay was first developed using analkyl amine fluoro-surfactant in the wash solution. The wash solutionwas used as a probe purge after delivery of the assay reactants and alsoas a washing solution. In order to substitute the wash solutionsurfactant with a more environmentally friendly hydrocarbon surfactant,the present invention was developed. The composition of the presentinvention can contain, but is not limited to, proteins, surfactants,buffer ions, and salt compositions that do not significantly upset theequilibrium of free non-protein bound and protein bound portions ofthyroxine in the serum or plasma. Thyroxine binding proteins arecomprised of albumin, thyroxine binding globulin, and transthyretin.

In one embodiment of the present invention, EHS is the stabilizing agentand is titrated within an optimized concentration range, in the case forFT4 between about 0.002% volume/volume and about 0.015% volume/volume,preferably at 0.004% volume/volume (or 181 micromolar) in the finalreaction composition, to aid in the maintenance of the free to boundequilibrium.

The following examples of how to use the present invention may set forthone or more, but not all exemplary embodiments of the present inventionas contemplated by the inventors, and thus, are not intended to limitthe present invention and the scope of the claims in any way.

EXAMPLES

The Access Free T4 assay (BCD) is a two-step enzyme immunoassay.Monoclonal anti-Thyroxine (T4) antibody (BCI) coupled to biotin, abiological sample containing T4, buffered protein solution containingstabilizing agent, and streptavidin-coated solid phase are added to areaction vessel. During this first incubation the anti-T4 antibodycoupled to biotin binds to the solid phase and the free T4 in thesample. After incubation in a reaction vessel, materials bound to thesolid phase are held in a magnetic field while unbound materials arewashed away. Next, buffered protein solution and triiodothyronine(T3)-alkaline phosphatase conjugate are added to the reaction vessel.

The T3-alkaline phosphatase conjugate binds to the vacant anti-T4antibody binding sites. After incubation in a reaction vessel, materialsbound to the solid phase are held in a magnetic field while unboundmaterials are washed away. Then, the chemiluminescent substrateLumi-Phos®530 (Lumigen Inc., Southfield, Mich.) is added to the vesseland light generated by the reaction is measured with a luminometer. Thelight production is inversely proportional to the concentration of freeT4 in the sample. The amount of analyte in the sample is determined froma stored, multi-point calibration curve. Serum and plasma (heparin) arethe recommended samples.

BCI's Access Free T4 A33070A assay kit includes an Access Free T4Reagent Pack Cat. No. 33880: 100 determinations, 2 packs, 50 tests/pack.It is provided ready to use. The reagent pack contains the followingReagents A through E:

Reagent A: Dynabeads® paramagnetic particles coated with streptavidin ina TRIS buffer with protein (aves), surfactant, 0.125% NaN₃, and 0.125%ProClin® 300 (available from Rohm and Haas, Philadelphia, Pa.).

Reagent B: TRIS buffered saline with protein (aves), surfactant, <0.1%NaN₃, and 0.1% ProClin 300.

Reagent C: TRIS buffered saline with protein (aves), surfactant, 0.125%NaN₃, and 0.125% ProClin 300.

Reagent D: Triiodothyronine-alkaline phosphatase (bovine) conjugate in aTRIS buffer with protein (aves), surfactant, <0.1% NaN₃, and 0.1%ProClin 300.

Reagent E: Mouse monoclonal anti-Thyroxine (T4) coupled to biotin in aTRIS buffer with protein (aves and murine), surfactant and stabilizingagent, 0.125% NaN₃, and 0.125% ProClin® 300.

An additional reagent, not part of the reagent pack itself and used forconvenience in screening numerous stabilizing agents, is designated asVial Reagent F. Vial Reagent F is a buffer containing at least onestabilizing agent (for example, sulfonated surfactants as referenced inFIG. 3 or FIG. 4).

Example 1 Stabilizing Agent Only in Reagent E of Reagent Pack

The first reaction comprises the following sequential additions to thereaction vessel:

-   -   1) 50 uL of Reagent E (containing stabilizing agent, e.g.,        0.016% EHS);    -   2) 30 uL of analyte-containing sample;    -   3) 30 uL of system wash buffer (containing no stabilizing        agent);    -   4) 30 uL of Reagent B (containing no stabilizing agent);    -   5) 50 uL of Reagent A.        -   Following incubation and washes of the reaction vessel, the            second reaction comprises the following sequential additions            to the reaction vessel:    -   6) 220 uL of Reagent C;    -   7) 50 uL of Reagent D;    -   8) 80 uL of system wash buffer (containing no stabilizing        agent).        -   Following incubation and washes of the reaction vessel, the            concentration of the free analyte in the sample is measured            using the detection system.

Example 2 Stabilizing Agent Only in Reagent B of Reagent Pack

The first reaction comprises the following sequential additions to thereaction vessel:

-   -   1) 50 uL of Reagent E (containing no stabilizing agent);    -   2) 30 uL of analyte-containing sample;    -   3) 30 uL of system wash buffer (containing no stabilizing        agent);    -   4) 30 uL of Reagent B (containing stabilizing agent, e.g.,        0.016% EHS);    -   5) 50 uL of Reagent A.        -   Following incubation and washes of the reaction vessel, the            second reaction comprises the following sequential additions            to the reaction vessel:    -   6) 220 uL of Reagent C;    -   7) 50 uL of Reagent D;    -   8) 80 uL of system wash buffer (containing no stabilizing        agent).        -   Following incubation and washes of the reaction vessel, the            concentration of the free analyte in the sample is measured            using the detection system.

Example 3 Stabilizing Agent Only in Vial Reagent F of Vial System

The first reaction comprises the following sequential additions to thereaction vessel:

-   -   1) 50 uL of Reagent E (containing no stabilizing agent);    -   2) 30 uL of analyte-containing sample;    -   3) 30 uL of Vial Reagent F (containing wash buffer with at least        one of the stabilizing agents referenced in FIG. 3 or FIG. 4);    -   4) 30 uL of Reagent B (containing no stabilizing agent);    -   5) 50 uL of Reagent A.        -   Following incubation and washes of the reaction vessel, the            second reaction comprises the following sequential additions            to the reaction vessel:    -   6) 220 uL of Reagent C;    -   7) 50 uL of Reagent D;    -   8) 80 uL of Vial Reagent F (containing wash buffer with at least        one of the stabilizing agents referenced in FIG. 3 or FIG. 4).        -   Following incubation and washes of the reaction vessel, the            concentration of the free analyte in the sample is measured            using the detection system.

Example 4 Stabilizing Agent Only in System Wash Buffer

The first reaction comprises the following sequential additions to thereaction vessel:

-   -   1) 50 uL of Reagent E (containing no stabilizing agent);    -   2) 30 uL of analyte-containing sample;    -   3) 30 uL of system wash buffer in a salt matrix such as 20 mM        TRIS, 0.15M NaCl, 0.1% Proclin 300, 0.1% NaN3, pH 8.0,        containing at least one stabilizing agent such as EHS or other        sulfonated surfactants as referenced in FIG. 3 or FIG. 4;    -   4) 30 uL of Reagent B (containing no stabilizing agent);    -   5) 50 uL of Reagent A.        -   Following incubation and washes of the reaction vessel, the            second reaction comprises the following sequential additions            to the reaction vessel:    -   6) 220 uL of Reagent C;    -   7) 50 uL of Reagent D;    -   8) 80 uL of system wash buffer in a salt matrix such as 20 mM        TRIS, 0.15M NaCl, 0.1% Proclin 300, 0.1% NaN3, pH 8.0,        containing at least one stabilizing agent such as EHS or other        sulfonated surfactants as referenced in FIG. 3 or FIG. 4.        -   Following incubation and washes of the reaction vessel, the            concentration of the free analyte in the sample is measured            using the detection system.

These examples illustrate possible embodiments of the present invention.While the invention has been particularly shown and described withreference to some embodiments thereof, it will be understood that theyhave been presented by way of example only, and not limitation, andvarious changes in form and details can be made therein withoutdeparting from the spirit and scope of the invention. Thus, the breadthand scope of the present invention should not be limited by any of theabove-described exemplary embodiments, but should be defined only inaccordance with the following claims and their equivalents.

All documents cited herein, including journal articles or abstracts,published or corresponding U.S. or foreign patent applications, issuedor foreign patents, or any other documents, are each entirelyincorporated by reference herein, including all data, tables, figures,and text presented in the cited documents.

1. A method for measuring a concentration of a free analyte, whereinanalyte exists in an equilibrium between free analyte and bound analytein a sample, the method comprising: (a) forming a test mixture by addingto a vessel: (i) a capture ligand for the free analyte, (ii)2-ethyl-hexyl sulfate, or salt thereof in an amount effective topreserve an equilibrium between free analyte and bound analyte, and(iii) the sample comprising the analyte; (b) adding a detection systemto the vessel; and (c) measuring the concentration of the analyte in thesample using the detection system.
 2. The method of claim 1, wherein theanalyte is a thyroid hormone.
 3. The method of claim 1, wherein thecapture ligand is analyte-specific and selected from the groupconsisting of antibody, antibody fragment, antibody mimic,analyte-specific binding protein, and combinations thereof.
 4. Themethod of claim 3, wherein the analyte-specific binding protein isintrinsic factor or folate-binding protein.
 5. The method of claim 1,wherein the detection system comprises a solid phase coated with acapture ligand-specific binding molecule, a detectable label, orcombination thereof.
 6. The method of claim 5, wherein the solid phaseis a paramagnetic particle.
 7. The method of claim 5, wherein thecapture ligand-specific binding molecule is hapten-specific, and whereinthe capture ligand is haptenated.
 8. The method of claim 7, wherein thehapten is biotin.
 9. The method of claim 5, wherein the captureligand-specific binding molecule is selected from the group consistingof streptavidin, avidin, biotin-specific antibody, biotin-specificantibody fragment, and biotin-specific antibody mimic.
 10. The method ofclaim 1, wherein the capture ligand is immobilized on a solid phase. 11.The method of claim 1, wherein a concentration of 2-ethyl-hexyl sulfatein the test mixture is about 5 micromolar to about 750 micromolar. 12.The method of claim 1, wherein a concentration of the 2-ethyl-hexylsulfate in the test mixture is about 75 micromolar to about 650micromolar.